Synthesis and evaluation of novel phosphoramidate prodrugs of 2′-methyl cytidine as inhibitors of hepatitis c virus NS5B polymerase

Article abstract of DOI:10.1016/j.bmcl.2009.01.035

A variety of new prodrugs of 2′-methyl cytidine based on acyloxy ethylamino phosphoramidates have been synthesized and tested in vitro and in vivo for their biological activity. Compared with the parent drug a 10- to 20-fold increase in formation of nucle

Full text of DOI:10.1016/j.bmcl.2009.01.035

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1392–1395
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of novel phosphoramidate prodrugs of 20-methyl
cytidine as inhibitors of hepatitis c virus NS5B polymerase
a
a
a
a
Monica Donghi a, , Barbara Attenni , Cristina Gardelli , Annalise Di Marco , Fabrizio Fiore ,
*
Claudio Giuliano a, Ralph Laufer a, Joseph F. Leone b, Vincenzo Pucci a, Michael Rowley a, Frank Narjes a
a Department of Medicinal Chemistry, IRBM-MRL Rome, Via Pontina Km 30,600, 00040 Pomezia (Rome), Italy
b Department of Preparative Chemistry & Separation, MRL Rahway, 126 E. Lincoln Avenue, PO BOX 2000, 07065-0900, NJ, USA
a r t i c l e i n f o
a b s t r a c t
A variety of new prodrugs of 20-methyl cytidine based on acyloxy ethylamino phosphoramidates have
been synthesized and tested in vitro and in vivo for their biological activity. Compared with the parent
drug a 10- to 20-fold increase in formation of nucleotide triphosphate in rat and human hepatocytes
could be achieved.
Article history:
Received 24 November 2008
Revised 9 January 2009
Accepted 14 January 2009
Available online 19 January 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Antiviral
HCV polymerase
Prodrugs
Phosphoramidates
Triphosphate
Hepatitis C virus (HCV) infection is a serious worldwide health
problem affecting about 2% of the world population (WHO data).1
While the early stages of the disease are typically asymptomatic,
the majority of HCV infections progress to chronic infection, which
is associated with an increased risk of cirrhosis, hepatocellular car-
cinoma, and liver failure. At present, the standard of care available
to patients with chronic HCV infection is a combination of ribavirin
and interferon-based therapies, which leads to a sustained viro-
logic response in only about half of the patients treated.2
Efforts to discover more effective drugs to treat HCV-infected
patients have focused on several possible targets, including the
NS5B RNA-dependent RNA polymerase.3 20-Methyl cytidine4 has
been shown to be an inhibitor of hepatitis virus C NS5B in cell cul-
ture.5 The 50-triphosphate of this nucleoside is a potent active site
inhibitor of hepatitis virus C polymerase.6 To enhance the bioavail-
ability of 20-methyl cytidine its 30-valine ester has been prepared
(NM283 = Valopicitabine 1, Fig. 1)7 and a 1.2 log10 viral reduction
in HCV RNA was observed in patients upon dosing 800 mg qd.8
Its development has been stopped in phase II due to an unfavorable
risk/benefit profile observed in clinical testing.3c
phorylation of the nucleoside to its monophosphate (NMP). To cir-
cumvent this problem, a suitable prodrug strategy would be one
aimed at the delivery of the nucleoside monophosphate inside
the target cell.11
To this end, different approaches have been described in the lit-
erature. For example, Metabasis Therapeutics has reported cyclic
cytochrome P450-3A-cleavable phosphate esters as liver tar-
geted-prodrugs (so-called HepDirect prodrugs).12 Furthermore, in
the antiviral field, McGuigan and co-workers have described aryl-
oxy phosphoramidates (incorporating an amino acid ester) as suit-
able pro-moieties, specifically to improve delivery of AZT and d4T
(ProTide approach).13
In the search for HCV NS5B polymerase inhibitors we applied an
analogous phosphoramidate approach to 20-methyl cytidine.14 As a
variation on the theme we also evaluated replacing the amino acid
moiety with 2-aminoethanol. Although Imbach et al. reported this
NH2
N
For a variety of nucleosides with modified core structure (e.g.,
d4T or ara-C) a poor turnover to the nucleoside triphosphate
(NTP) has been observed.9,10 In some cases this is attributed to
the inability of the nucleoside kinase to catalyze the initial phos-
HO
O
N
O
O
O
OH
1
NH2
* Corresponding author. Fax: +39 06 91093225.
E-mail address: monica_donghi@merck.com (M. Donghi).
Figure 1. Valopicitabine, NM283.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.035

M. Donghi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1392–1395
1393
Benzoyl and acetyl esters were prepared (examples 6–7). These
compounds exhibited encouraging improvements in cell-based
activity, when compared with NM283. A significant step forward
in terms of activity could then be made with the introduction of
branched chain alkanoic esters 8–12 (Table 1). We found that not
only small esters like those based on iso-butanoic acid were toler-
ated, but also longer chain esters like 2-ethylbutanoates and 2-pro-
pylpentanoates gave rise to very active compounds in the cell-
based assay.
NH2
N
O
O P
R4
R3
N
O
R1
N
O
O
HO
OH
O
O
R2
Figure 2. Acyloxyethylamino phosphoramidate prodrugs.
Substitution of the ethanolamine (13–19) was at best tolerated
(16b) but still compatible with sub-lM levels of inhibition in the
approach failed in their study on AZT derivatives,15 we were
pleased to find that this strategy proved successful for our sub-
strate. Herein we report the design, synthesis and biological evalu-
ation of this novel 20-Me-C-based series (Fig. 2).
replicon. However, simple N-methylation of the ethanolamine
nitrogen (R4 = Me, 20) or exchanging the ethanolamine moiety
with 3-hydroxy-prolinol (example 21) completely abrogated
activity.
The synthesis of the compounds was first performed under clas-
sical conditions by reaction of the unprotected nucleoside with a
phosphoramide chloride. This approach suffered from two major
liabilities. The first is a physical issue related to the limited solubil-
ity of 20-Me-C in organic solvents. The second derives from the
poor yield in the reaction of the amino alcohol fragments with phe-
nyl phosphorodichloridate. While the latter reacted smoothly with
amino acid derivatives, only poor results were observed with ami-
no alcohol derivatives. To circumvent these issues, a different route
was developed. We found that the compounds could be conve-
niently prepared by first masking the secondary and tertiary alco-
hols on the nucleoside 2 as a 20,30-acetonide (Scheme 1).
Due to the chirality of the phosphorus all compounds were ob-
tained as a mixture of diastereomers. These could be separated by
HPLC or SFC. In most cases the second eluting diastereomer (e.g.,
6b, 9b, 10b and 16b) turned out to be more active in the replicon
assay than the first eluting product although in the end both dia-
stereomers should yield the same nucleotide. Thus, a plausible
hypothesis for pro-moiety activation would be via a stereoselective
enzymatic first step (as for the McGuigan prodrug), whereby this
stereochemical preference is then reflected in the replicon activity.
The data reported in Table 1 refer to the second eluted diastereo-
isomer if not otherwise denoted.
For representative compounds the actual formation of nucleo-
side triphosphate from the prodrug was studied in rat and human
hepatocytes, (Table 2). We were very pleased to observe that the
replicon data could be associated with levels of NTP formation in
comparison with the reference compound NM283. Compounds
which had been found to be inactive in the replicon assay also
did not give any appreciable level of NTP formation in hepatocytes
(e.g., 20b). It should be noted that the difference in activity be-
tween the first and the second eluting diastereomers observed in
the replicon assay could not be fully explained by the hepatocyte
assay data. However, the interspecies variability observed in the
hepatocytes would be in agreement with an enzymatic activation.
Taking into consideration the replicon and hepatocyte data we
identified 10, 11, 12 and 16 as the most promising compounds.
Though 11 and 16 showed very satisfactory NTP formation in
human hepatocytes, we considered the release of one equivalent
of pivalic acid or tryptanol per molecule as a potential liability
from a toxicity point of view,18 which led us to prefer compounds
10 and 12.
The resulting 50-unprotected nucleoside 3 was then reacted
with diphenyl phosphite to give the common intermediate 4. This
material was not isolated but subjected directly to an Atherton-
Todd reaction with an O-acyl aminoethanol in the presence of tet-
rachloromethane.19 Careful deprotection of the phosphoramidates
5 afforded the desired prodrugs 6–19 in moderate overall yield
(25% from nucleoside 2). Separation of the two diastereoisomers
was achieved for each derivative by reversed phase HPLC.
All compounds were routinely tested in the replicon assay.16 For
representative compounds the formation of nucleoside triphos-
phate was assessed in screening mode in human and rat hepato-
cytes.17 Those compounds showing promising results were then
profiled further.
In this report, we focus on the structure–activity relationship
around the amino alcohol moiety, with the phenoxy functionality
on the phosphoramidate remaining unvaried. In particular, initial
studies were directed towards the preparation of a series of esters
of unsubstituted ethanolamine (R1 = R3 = H).
NH2
NH2
NH2
N
O
(PhO)2P(=O)H,
py
p-TsOH,
N
N
O
O P
H O
acetone
HO
N
HO
N
N
O
O
O
O
O
MeO
OMe
HO
OH
O
O
O
O
80%
2
3
4
O
R3
NH2
N
NH2
N
NH2 . HCl
R1
R2
O
O
O
TFA, H2O
80%
O P
O P
HN
O
R1
N
HN
O
R1
N
O
O
NEt3, CCl4,
THF
O
O
R3
R3
O
O
HO
OH
O
O
40%
O
O
R2
5
R2
6-19
Scheme 1. Preparation of new amino alcohol based arylphosphoramidate diester prodrugs.20

1394
M. Donghi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1392–1395
Table 1
In vitro activity
Compound*
R1
R2
R3
R4
Replicon (10% FCS) nM
NM283
6b
7b
–
H
H
H
H
H
H
H
H
H
H
Me
Bn
–
Ph
Me
cHep
3-Pentyl
3-Pentyl
4-Hep
4-Hep
tBu
iPr
iPr
iPr
iPr
–
–
7600 (n = 29)
1960
1250
1160
2830
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
8b
9 (1st)
9b (2nd)
10a (1st)
10b (2nd)
11b
12b
13b
14b
15b
625
450 (n = 4)
240 (n = 3)
226
182
585
542
921
16a (1st)
iPr
iPr
H
H
H
H
717
218
N
H
16b (2nd)
N
H
17b
18b
19b
20b
iBu
Bn
nBu
H
tBu
tBu
tBu
iPr
H
H
H
H
H
H
H
Me
1840
840
659
>10000
NH2
N
O
O P
N
O
N
O
–
–
–
–
>20000
O
21b
O
HO OH
O
R substituents refer to Figure 2.
*
b: Second eluting diastereomer if not noted otherwise.
Kinetics of NTP formation of 10b was further evaluated in hepa-
tocytes from different species (Table 3) which confirmed the data
obtained in screening mode.
Table 2
Stability in human, dog and rat plasma over 120 min
Compound
NTP levela, (
Rat hepatocytes
<14
l
M), 2 h (n = 2)
% of initial @ 120 min
human/dog/rat
To further profile the prodrugs, levels of NTP formation in rat li-
ver after oral administration were measured. As illustrated for 6,
10b and 19, in general NTP formation was very low (Table 4) with
no improvement over NM283. To assess whether the disappointing
liver levels might be related to poor intestinal absorption, the per-
meability of selected prodrugs across monolayers of Caco-2 cells
was determined. Most compounds tested had very low cell perme-
ability and displayed active efflux in the basolateral-apical direc-
tion (data not shown). Interestingly, although still poorly
permeable, compound 19 did not show significant efflux (perme-
ability coefficient = 1.40 cm/s ꢀ 106; BA/AB ratio = 1.7). Thus, de-
spite the presence of the undesirable pivaloate group, it was used
as a model compound and dosed orally in rat. Unfortunately, as
mentioned above, no improvement in NTP formation in liver was
observed (Table 4), suggesting active efflux of the parent prodrug
is not the only factor limiting liver NTP levels. At this point alterna-
tive routes of administration were evaluated and compound 10b
Human hepatocytes
NM283
6b
17 10
22 0.1
41
2
0/98/41
7b
178 64
207 190
133 117
149 57
48
36 20
23
120 10
91 13
3
10a
10b
11b
12b
14b
15b
16a
16b
18b
19b
20b
100/100/100
100/100/100
2
0/95/15
0/36/3
83/90/0
33
8
26
6
104 20
127
62
2
6
128 115
191 16
33 13
140 21
97 29
<14
100/100/100
7
1
a
Incubated with 10
l
M compound.
was dosed subcutaneously in rat (1.5 lmol/kg). Gratifyingly high
liver NTP levels were observed at 6 h. This is in stark contrast to
NM283 and demonstrated the potential of our strategy.
In conclusion, modifying McGuigan’s approach we have identified a
novel class of phosphoramidates starting from the known HCV poly-
merase inhibitor NM283. The prodrugs we identified have good po-
The stability in rat, dog and human plasma was determined for
selected compounds (Table 2). When compared with other esters,
the 2-propylpentanoate esters 10a and 10b exhibited good stabil-
ity which was equaled only by the pivaloate ester 19. For 10b
essentially no degradation was observed over 120 min while 12b
was not stable in human and rat plasma.

M. Donghi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1392–1395
1395
Table 3
Van Poelje, P. D.; MacKenna, D. A.; Colby, T. J.; Montag, A. C.; Fujitaki, J. M.;
Linemeyer, D. L.; Bullough, D. A. J. Pharm. Exp. Ther. 2005, 312, 554; (d) Reddy,
K. R.; Boyer, S. H.; Erion, M. D. Tetrahedron Lett. 2005, 46, 4321; (e) Boyer, S. H.;
Sun, Z.; Jiang, H.; Esterbrook, J.; Gomez-Galeno, J. E.; Craigo, W.; Reddy, K. R.;
Ugarkar, B. G.; MacKenna, D. A.; Erion, M. D. J. Med. Chem. 2006, 49, 7711; (f)
Erion, M. D.; Reddy, K. R.; MacCoss, M.; Olsen, D. B. International Patent
Application WO 2007022073 A2, 2007.; (g) Hecker, S. J.; Reddy, K. R.; van
Poelje, P. D.; Sun, Z.; Huang, W.; Varkhedkar, V.; Reddy, M. V.; Fujitaki, J. M.;
Olsen, D. B.; Koeplinger, K. A.; Boyer, S. H.; Linemeyer, D. L.; MacCoss, M.; Erion,
M. D. J. Med. Chem. 2007, 50, 3891.
NTP formation in hepatocytes (AUC0ꢁ4 h
,
l
M h)a
Human
315
Compound
Rat
Dog
50
Rhesus
310
Rabbit
59
10b
2770
a
Incubated with 10
l
M compound.
Table 4
NTP levels in rat liver 6 h after oral dosing
13. (a) McGuigan, C.; Cahard, D.; Sheeka, H. M.; De Clercq, E.; Balzarini, J. Bioorg.
Med. Chem. Lett. 1996, 6, 1183; (b) McGuigan, C.; Cahard, D.; Sheeka, H. M.; De
Clercq, E.; Balzarini, J. J. Med. Chem. 1996, 39, 1748; (c) Balzarini, J.; Kruining, J.;
Wedgwood, O.; Pannecouque, C.; Aquaro, S.; Perno, C. F.; Naesens, L.;
Witvrouw, M.; Heitjink, R.; De Clercq, E.; McGuigan, C. FEBS Lett. 1997, 410,
324; (d) Siddiqui, A.; McGuigan, C.; Ballatore, C.; Srinivasan, S.; De Clercq, E.;
Balzarini, J. Bioorg. Med. Chem. Lett. 2000, 10, 381.
14. Gardelli, C. et al., in preparation.
15. Egron, D.; Lefebvre, I.; Périgaud, C.; Beltran, T.; Pompon, A.; Gosselin, G.;
Aubertin, A.-M.; Imbach, J.-L. Bioorg. Med. Chem. Lett 1998, 8, 1045.
16. (a) Lohmann, V.; Korner, F.; Koch, J.-O.; Herian, U.; Theilmann, L.;
Bartenschlager, R. Science 1999, 285, 110; (b) Bartenschlager, R.; Lohmann, V.
Antiviral Res. 2001, 52, 1; (c) De Francesco, R.; Migliaccio, G.; Paonessa, G.
International Patent Application WO 2002059321 A2, 2002.
17. (a) Di Marco, A. et al., in preparation.; Human (pool of 10 donors) and rabbit
hepatocytes were obtained from In Vitro Technologies (IVT), rhesus hepatocytes
from Cambrex Biosciences while rat and dog hepatocytes were prepared as
described in (b) Giuliano, C.; Fiore, F.; Di Marco, A.; Padron Velazquez, J.; Bishop,
A.; Bonelli, F.; Gonzalez-Paz, O.; Marcucci, I.; Harper, S.; Narjes, F.; Pacini, B.;
Monteagudo, E.; Migliaccio, G.; Rowley, M.; Laufer, R. Xenobiotica 2005, 35, 1035.
18. Brass, E. P. Pharmacol. Rev. 2002, 54, 589.
Compound
R1
R2
R3
Rat liver NTP level, (nmol/g), 6 h
p.o. (30
l
mol/kg)
s.c. (1.5
BLQ
lmol/kg)
NM283
6
10b (2nd)
19
–
H
H
–
Ph
4-Hep
tBu
–
0.2
0.3
0.2
0.2
H
H
H
7.1
nBu
BLQ = 0.1 nmol/g.
tency in the replicon assay. Furthermore, we were able to demonstrate
that these newprodrugs formhigher levelsof triphosphate than NM283
in hepatocytes (human and preclinical species) and in rat liver after s.c.
dosing. However, these promising results did not translate into the for-
mation of high liver NTP levels in rat after p.o. dosing. Further studies
addressing oral bioavailability, mechanism of pro-moiety activation
and conversion to NTP ongoing.
19. (a) Atherton, F. R.; Openshaw, H. T.; Todd, A. R. J. Chem. Soc. 1945, 660; (b)
Gamble, M. P.; Smith, A. R. C.; Wills, M. J. Org. Chem. 1998, 63, 6068; (c) Pettit,
G. R.; Anderson, C. R.; Gapud, E. J.; Jung, M. K.; Knight, J. C.; Hamel, E.; Pettit, R.
K. J. Nat. Prod. 2005, 68, 1191.
Acknowledgments
20. Synthetic and brief spectroscopic data on compound 10a,b: Step 1: 20-C-
Methylcytidine (19.4 mmol) was dissolved in acetone (0.04 M) and p-
toluenesulfonic acid (23.3 mmol, 1.2 equiv) and 2,2-dimethoxypropane
(194 mmol, 10 equiv) were added. The resulting slurry was stirred for 24 h at
RT. The solvent was evaporated, the residue was dissolved in MeOH and
Amberlite A-26 (previously washed with 2 N NaOH and H2O) was added. The
resulting mixture was stirred for 2 h. The Amberlite was filtered off and the
filtrate was evaporated. The resulting crude product was purified by column
chromatography on silica gel (DCM/MeOH = 9:1) to give the desired product as a
colorless powder. 1H NMR (300 MHz, CD3OD) d 7.96 (d, J = 7.56 Hz, 1H), 6.18 (s,
1H), 5.90 (d, J = 7.56 Hz, 1H), 4.51–4.48 (m, 1H), 4.28–4.23 (m, 1H), 3.86 (dd,
J = 3.04 and 12.12 Hz, 1H), 3.78 (dd, J = 3.52 and 12.12 Hz, 1H), 1.59 (s, 3H), 1.43
(s, 3H), 1.25 (s, 3H); Mass found for C13H19N3O5: MS (ES+) m/z 298.5 (M+H)+.
Step 2: 20-C-Methyl-20,30-O-(1-methylethylidene)-cytidine (4.37 mmol) was
dissolved in pyridine (1.45 M) in presence of molecular sieves. The
resulting solution was cooled to 0 °C, diphenyl phosphite (80%, 5.68 mmol,
1.3 equiv) was added, and the mixture was stirred for 1 h at 0 °C. The
solvent was evaporated and the residue dissolved in THF/CCl4 (8:1, 0.12 M).
The resulting solution was cooled to 0 °C and Et3 N (26.2 mmol, 6.0 equiv),
and a solution of aminoethyl 2-propylpentanoate hydrochloride (5.68 mmol,
1.3 equiv) in iPrOH/THF (1:1, 8 ml) were added. The mixture was stirred for
30 min at 0 °C and then was quenched by the addition of water. The
aqueous phase was extracted three times with EtOAc, the combined organic
phases were washed with brine and dried over Na2SO4. All volatiles were
evaporated in vacuo. The crude product was purified by column
chromatography on silica gel (DCM/MeOH = 95:5) to give a colorless solid
as mixture of diastereoisomers which was used without further
characterization. Mass found for C29H43N4O9P: MS (ES+) m/z 623.6 (M+H)+.
This material was dissolved in a solution of TFA/H2O (8:2, 0.098 M) and the
resulting solution was warmed to 30 °C and stirred for 20 min. The solvent
was evaporated and the residue was dissolved in acetonitrile and purified by
The authors thank Renzo Bazzo for NMR studies, Alessandra
Ceccacci, Nadia Gennari and Sergio Altamura for carrying out the
biological assays, and Isabella Marcucci and Antonella Cellucci for
carrying out the NTP formation in hepatocytes.
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lm, 21.20 ꢀ 250 mm.
Mobile phase: acetonitrile/H2O buffered with 5 mM AMBIC). The fractions
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afford the title compounds as colorless powders.
First-eluting diastereoisomer 10a1
H NMR (300 MHz, CD3OD) d 7.71 (d,
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J = 7.6 Hz, 1H), 7.43–7.19 (m, 5H), 6.07 (s, 1H), 5.83 (d, J = 6.8 Hz, 1H), 4.58–
4.5 (m, 1H), 4.47–4.34 (m, 1H), 4.18–4.05 (m, 3H), 3.74 (d, J = 9.1 Hz, 1H),
3.35–3.2 (m, 2H), 2.48–2.33 (m, 1H), 1.66–1.37 (m, 4H), 1.37–1.23 (m, 4H),
1.1 (s, 3H), 0.95–0.86 (m, 6H). 31P NMR: (300 MHz CD3OD) d: 5.45; Mass
found for C26H39N4O9P: MS (ES+) m/z 583.4 (M+H)+
.
Second-eluting diastereoisomer 10b1
H NMR (300 MHz, CD3OD) d 7.71 (d,
J = 7.4 Hz, 1H), 7.46–7.37 (m, 2H), 7.33–7.21 (m, 3H), 6.05 (s, 1H), 5.81 (d,
J = 7.3 Hz, 1H), 4.56–4.50 (m, 1H), 4.44–4.34 (m, 1H), 4.18–4.05 (m, 3H),
3.76 (d, J = 9.1 Hz, 1H), 3.35–3.20 (m, 2H), 2.46–2.35 (m, 1H), 1.66–1.37
(m, 4H), 1.37–1.22 (m, 4H), 1.12 (s, 3H), 0.97–0.86 (m, 6H). 31P NMR:
(300 MHz CD3OD) d: 5.52. Mass found for C26H39N4O9P: MS (ES+) m/z
583.4 (M+H)+.